Project Summary/Abstract Our long term objective is to understand the differentiation of specialized cell types in the pancreas. Our general strategy is to study the beta-cell as a model to decipher the transcriptional network functioning in pancreatic progenitor cells and guiding their differentiation into specific mature cell types. Our Specific Aims for this funding period are directed toward understanding the role of a single factor, Nkx2.2, in this transcriptional network. Nkx2.2 plays a critical role in beta-cell differentiation and is uniquely expressed in early pancreatic progenitor cells, islet progenitor cells, and mature beta-cells, via distinct mechanisms in each cell type, making it a useful tool to understand how gene expression is differentially regulated in these related cell types. Our Aims are directed at two basic questions: Question 1: What distinguishes pancreatic progenitor cells at different stages of development and determines their ability to generate different cell types? Aim 1. Establish the difference in gene expression networks between early and late pancreatic progenitor cells. We will use sox9-GFP mice to sort the progenitor cells from different ages and to compare their gene expression patterns and networks. The information generated will be used to understand the requirements for generating beta-cells from progenitor cells, for identifying target genes of Nkx2.2 in early pancreatic progenitor cells, and for understanding what controls the activity of the Nkx2.2 1B promoter (Aim 2). Aim 2. Determine the mechanisms that direct expression of the Nkx2.2 1B promoter to the early progenitor cells. Using information from Aim 1, we will determine how the 1B promoter is activated in the early pancreatic progenitor cells. We will use mouse models to map the nkx2.2 1b promoter and to test the role of putative regulators. Question 2: How does Nkx2.2 regulate downstream genes? Aim 3. Test the role of Nkx2.2 in the translation of Nkx6.1. In the absence of Nkx2.2, Nkx6.1 protein levels in the fetal pancreas drastically decline after the secondary transition, but mRNA levels do not. We will test the role of the nkx6.1 5'and 3'UTR in this translational regulation by Nkx2.2, and the role of Nkx2.2 in the regulation of the Nkx6.1 IRES sequences. Aim 4. Determine how Nkx2.2 silences pHox2b expression. Islet Nkx2.2 and neural-crest pHox2B form a non-cell-autonomous negative feedback loop that regulates beta-cell proliferation. We will test the role of Nkx2.2-regulated islet signals in silencing pHox2B expression in pancreatic neural-crest cells. These studies will help explain how the differentiated state is established and maintained. Ultimately this information will help guide efforts to generate beta-cells for patients with diabetes.